Influence of sex on renin‐angiotensin‐aldosterone system metabolites and enzymes in Doberman Pinschers

Abstract Background Estrogen modulates the renin‐angiotensin‐aldosterone system (RAAS) in women, but sex differences have not been fully explored in dogs. Objective We hypothesized that the RAAS profile of intact female (IF) Doberman Pinschers (DP) would differ from spayed female (SF) and intact male (IM) DP. Animals Eighteen healthy DP (6 IF, 6 SF, 6 IM). Methods Absolute and indexed RAAS metabolites, angiotensin‐converting enzyme (ACE) and ACE2 activities, and genotypes (pyruvate kinase dehydrogenase 4, titin, and ACE variants) were compared among sex groups using Kruskal‐Wallis or chi‐square tests, and linear regression controlling for age. Data are expressed as median (minimum, maximum) and P < .05 was considered significant. Results The ACE activity was higher in IF DP (656 pmol/L; 436, 784) compared to SF DP (411 pmol/L; 287, 451; P = .01) and IM DP (365 pmol/L; 276, 1200; P = .04) after controlling for age. Angiotensin II, angiotensin I, and plasma renin activity marker (PRA‐S) were higher in IF DP compared to SF DP, but not significantly (P ≤ .25). After controlling for age, angiotensin 1‐7/angiotensin I was lower in IF DP compared to SF DP (P = .01). Genotypes did not differ among groups. Most DP (94%) were ACE variant positive. Conclusions and Clinical Significance Sex and reproductive status influenced the RAAS of DP, with IF DP showing genotype‐independent higher ACE activity. These findings hold implications for sterilization practices in female dogs, and support sex and reproductive status as a source of variability in RAAS studies. Additionally, the frequency of the ACE gene variant was very high in this group of DP.

cascade, angiotensin II, mediates classical pathway effects of vasoconstriction, sodium retention, aldosterone synthesis, and inflammation. 1 An alternative RAAS pathway counterbalances these actions of angiotensin II with vasodilatation, natriuresis, and anti-inflammatory effects through enhanced formation of angiotensin 1-7 from angiotensin I and II. 1 Estradiol is emerging as an important modulator of the RAAS in women, and the hormonal changes that occur during menopause appear to explain the increase in cardiovascular disease after the sixth decade. [3][4][5][6] Current research suggests that estradiol modulates various components of the RAAS to enhance angiotensin-mediated inflammation during infection or trauma, which may explain the better ability of women of child-bearing age to fight infections. 3 Once the stimulus is resolved, however, estradiol then acts as a switch to promote vasodilatation, natriuresis, and anti-inflammatory effects through the alternative RAAS pathway and activation of the angiotensin II receptor type 2. 3,5 Cascade changes resulting from estradiol influence in women are complex and include increases in angiotensinogen, angiotensin II receptor type 2 density, natriuretic peptides, and angiotensin 1-7, accompanied by decreases in renin concentration, angiotensin-converting enzyme (ACE) activity, angiotensin II receptor type I density, and aldosterone production. 6 Removal of the cardioprotective effect of estradiol with onset of menopause dysregulates the RAAS, resulting in unopposed classical pathway effects of vasoconstriction, sodium retention, and inflammation, such that the incidence of cardiovascular disease in postmenopausal women increases to results similar to those of men. 5,6 Sex differences in the RAAS cascade are largely unexplored in client-owned dogs aside from a study that showed an effect of breed, sex, and age on the urine aldosterone: creatinine ratio. 7 In that study, intact female (IF) dogs had significantly higher urine aldosterone: creatinine ratios than did spayed female (SF) dogs, intact male (IM) dogs, and neutered male dogs, but other aspects of the RAAS cascade were not evaluated. 7 The RAAS is increasingly recognized as a complex system that can be influenced by a variety of factors, including breed, sex, age, and genetic polymorphisms, many of which have not been explored. 6,[8][9][10][11] Understanding these confounding variables may help explain contradictory findings of previous studies of ACE inhibitors in dogs with heart disease, and is critical to the design of future studies. 7,[12][13][14] Additionally, knowledge about the impact of surgicallyinduced estrogen deficiency on the RAAS could be relevant in veterinary medicine.
Our purpose was to explore sex and female reproductive status as influential variables on the RAAS by evaluating circulating RAAS metabolites and enzyme activities in healthy, mature, IF, SF, and IM Doberman Pinscher (DP) dogs. We sought to comprehensively evaluate the circulating RAAS in these dogs using equilibrium dialysis to provide a global assessment of the cascade and build on previous work reporting urine aldosterone concentrations by ELISA methodology. 7 We hypothesized that the RAAS profile of IF DP would differ from that of SF and IM DP.

| METHODS
A convenience sample of 18 clinically healthy, mature DP (6 IF, 6 SF, 6 IM) was used. Residual serum from samples that were collected as part of an observational study evaluating the cardiac function of dogs eating grain-free and grain-inclusive diets 15 was used to evaluate core RAAS metabolites (angiotensin I, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-7, angiotensin 1-5, aldosterone) and the enzyme activities of ACE and ACE2. Dogs were genotyped for variants associated with dilated cardiomyopathy (DCM) in this breed, including DCM1 (pyruvate kinase dehydrogenase 4) and DCM2 (titin), 16 as well as for the known ACE gene variant 17,18 (https://cvm. ncsu.edu/nc-state-vet-hospital/small-animal/genetics/submit-dnatesting/). Sample collection was approved by the University of Florida IACUC committee (#201810504), and client consent was obtained from owners.
The inclusion of a single breed (DP) was intended to minimize variability that could be introduced by including dogs of multiple breeds.
Dogs were included in the study if they were eating a grain-inclusive diet, were between 18 and 96 months of age, and were apparently healthy based on history, physical examination, 20-minute ECG monitoring during echocardiography, and echocardiography results using breed-specific reference values. 19 The age range was chosen to avoid sexually immature and geriatric dogs. Medications other than heartworm prevention were not allowed. Intact female DP were not in estrus, not pregnant, and not lactating at the time of sampling. Dogs were fasted before blood collection between 10 AM and 2 PM and serum was removed immediately after centrifugation. Samples were stored at À80 C until batch analysis.

| Equilibrium dialysis to assess the RAAS
The equilibrium concentrations of 6 different angiotensin peptide metabolites (angiotensin I, angiotensin II, angiotensin III, angiotensin IV, angiotensin 1-5, angiotensin 1-7) and aldosterone were quantified by liquid chromatography-mass spectrometry/mass-spectroscopy (LC-MS/MS), performed by a service provider laboratory (Attoquant Diagnostics, Vienna, Austria), using previously validated and described methods. 14 Analyte concentrations were reported in pmol/L and the lower limit of quantification was reported for each metabolite. The activities of ACE and ACE2 were measured using a kinetics approach with spiked substrate. 20,21 Activity of ACE was determined by measuring the formation of angiotensin II after addition of angiotensin I in the presence and absence of an ACE inhibitor and a chymase inhibitor to determine the ACE-inhibitor sensitive fraction of angiotensin II generation. Activity of ACE2 was determined by measuring the formation of angiotensin 1-7 in the presence and absence of an ACE2 inhibitor using recombinant human ACE2 as a reference standard. The ratio of angiotensin II to angiotensin I was calculated as a marker for ACE activity (ACE-S). 14 Angiotensin I and angiotensin II were summed as a marker for plasma renin activity (PRA-S). 22 The ratio of aldosterone to angiotensin II (AA2) was calculated as a unitless indicator of adrenal responsiveness to angiotensin II stimulation of aldosterone release. 14 The sum of angiotensins 1-7 and 1-5 divided by the sum of angiotensins I, II, 1-7, and 1-5 was calculated as a unitless marker of renin-independent alternative RAAS activation (ALT-S). The angiotensin 1-7/angiotensin I ratio was calculated to account for overall RAAS activity and aid interpretation of the balance of angiotensin 1-7 formation and breakdown. This approach was necessary considering the high prevalence of the ACE gene variant in this group of dogs and the impact of overall RAAS activation on all downstream metabolites that result from metabolism of angiotensin I.

| Statistical analysis
Demographic data including genotype, weight, and age were reported for each sex group as median (minimum, maximum), except for genetic status which was reported as observed frequencies (n; %). A priori power analysis was not performed because the study was exploratory.
Proportions of genetically positive (homozygous or heterozygous, all variants) or negative (wildtype) dogs were compared among sex groups using a chi-square test. The distributions of age (months), body weight (kg), and RAAS metabolites, ratios, and enzymes were compared among sex groups using the nonparametric Kruskal-Wallis test followed by Dunn's test for multiple comparisons. Multiple linear regression was used to adjust the analysis for potential confounding effects of age on investigated associations among sex groups and RAAS variables (rank data). Using linear regression, dogs were assigned into 1 of 2 age groups based on the median distribution (i.e., 19 concentrations below the lower limit of quantification were entered as half of the lower limit of quantification for statistical comparisons.
P < .05 was considered significant.

| RESULTS
Most dogs (17/18) were ACE variant positive, whereas fewer dogs were positive for DCM1 (6/18) or DCM2 (7/18; Tables 1 and 2). Age was higher in IF DP and SF DP compared to IM DP but the difference among groups did not reach statistical significance (Table 2; P = .09).
Weight and the proportions of DCM1, DCM2, and ACE variant positive and negative DP were not significantly different among sex groups (P > .2).
In the univariable analysis (Table 2), ACE activity was different among sex groups, and it was highest in IF DP (P = .02). The angiotensin 1-7/angiotensin I ratio was lower in IF DP compared to SF DP or IM DP, but the difference among groups did not reach statistical significance (P = .06). Angiotensin I, PRA-S, and angiotensin II activity were higher in IF DP compared to SF DP, but the differences among groups were not significant (P ≤ .25). Finally, serum aldosterone concentrations were not different among sex groups (P = .67).
Using multivariable linear regression analysis, ACE activity was highest in IF DP compared to SF DP (P = .01) or IM DP (P = .04), after controlling for age. In addition, after controlling for age, the angiotensin 1-7/angiotensin I ratio was lower in IF DP compared to SF DP (P = .01), but no difference was found between IF DP and IM DP (P = .32). The remainder of the RAAS variables and ratios also were not different among sex groups before or after controlling for age.

| DISCUSSION
Sex and female reproductive status affected some components of the RAAS cascade in this small group of DP. The largest group differences were higher ACE activity in IF DP compared to SF DP and IM DP after controlling for age. These group differences were independent of the progesterone for the mineralocorticoid receptor could affect the relationship between serum and urine aldosterone concentrations. 6 Alternatively, our study may have been underpowered to detect differences among groups. Although we did not find sex group differences for aldosterone, we did find numerically (but not statistically) higher concentrations of other metabolites that are associated with classical RAAS activation in IF dogs compared to SF dogs (e.g., angiotensin I and II). Interestingly, the aldosterone concentrations for all sex groups in our study were notably higher than aldosterone concentrations reported for healthy purpose-bred dogs using the same methodology. 24 An influence of ACE variant positivity on aldosterone concentrations could have occurred in our study population, because nearly all DP had this variant, which is associated with a higher magnitude of aldosterone breakthrough after ACE inhibition in dogs with heart disease. 14 Other reasons for the differences among studies could be related to sampling, environment, or breed.
Although the frequency of the ACE variant in our dogs was very high, nearly all of the dogs were genetically positive and so this factor likely did not impact the sex group differences for ACE activity.
Despite different ACE activities, the surrogate marker for this enzyme (ACE-S), which is the ratio of angiotensin II to angiotensin I, was not different among groups. This seemingly discordant finding supports non-ACE generation of angiotensin II in the circulation, 14,25 although it was not directly measured in our study. Non-ACE enzymes, such as chymases, are capable of producing angiotensin II to maintain stable concentrations when ACE activity is low, thus explaining unaltered ACE-S, which represents the end result of angiotensin II formation relative to angiotensin I without consideration of the various pathways that produce it. The method we used to directly measure ACE activity is a better assessment of ACE activity than the surrogate ratio ACE-S because it accounts for chymase-generated angiotensin II by using both a chymase inhibitor and an ACE inhibitor to determine only the ACE inhibitor-sensitive fraction.
A previous study showed predispositions for the ACE variant in several breeds known to have genetically-based heart disease (e.g., Irish Wolfhounds, Cavalier King Charles Spaniels), but there were insufficient numbers of DP in that study to assess breed predisposition. 18 Our study suggests a high prevalence of the ACE variant in DP. The importance of this variant to genetically-based DCM in the DP requires study.
The lower angiotensin 1-7/angiotensin I ratio in IF DP compared to SF DP after controlling for age, indicates that there was relative classical RAAS predominance in IF DP and relative alternative RAAS predominance in SF DP. Relating angiotensin 1-7 to angiotensin I is important to account for overall RAAS influence, which can impact downstream metabolite formation globally. Interpretation of 1 metabolite without consideration for the others in the cascade could be misleading. The ALT-S calculation, which is an estimate of the entire alternative RAAS arm, was also lower in IF DP, although the differences among sex groups were not significant. The finding that angiotensin 1-7/angiotensin 1, but not ALT-S, was significantly lower in IF DP compared to SF DP could have been a consequence of small sample size or impact of the ACE variant. The major formation enzyme for the alternative RAAS (ACE2), was not different between groups, indicating that the generation of angiotensin 1-7 mediated by this enzyme was not different among groups. The ACE enzyme however also contributes to angiotensin 1-7 concentrations because it catalyzes the breakdown of angiotensin 1-7 to angiotensin 1-5, in addition to its well-known role of converting angiotensin I to angiotensin II. Therefore, lower ACE activity is expected to stabilize angiotensin 1-7 concentrations by decreased degradation.